RASLseq FASTQ reads to RASLprobe counts.

RASL-seq is a powerful and inexpensive method to assess gene expression without the need for RNA isolation1. We recently published a modified protocol using the RNA ligase Rnl2 which demonstrated dramatically increased ligation efficiency2. This python package offers an alignment method leveraging BLASTn, pandas, py-editdist, and NumPy. Optimizations will follow in the near future.

-f : absolute path to fastq file (accepts gzip files)

-p : absolute path to probes file

-w : absolute path to annotations file

-d : absolute path to write directory for blast database

-b : absolute path to blast bin directory

-o : absolute path of output file

-s : specifies sequencer id in fastq index line, e.g. @HISEQ

-P : verbose printing

example command:
python /path/to/RASLseqAnalysis.py -f /path/to/your.fastq.gz -s @HISEQ -p /paht/to/RASL.probes -w /path/to/annotations.bc -d /path/to/blastdb/write_dir/ -b /path/to/blast/ncbi-blast-2.2.26+/bin/ -P -o /path/to/output.txt

example data: can be found in the data directory

FASTQ: standard FASTQ format (optionally gzipped)

X.probes: tab-separated file describing the RASLseq Probes with the following columns and column headers

AcceptorProbeSequence
DonorProbeSequence
AcceptorAdaptorSequence
DonorAdaptorSequence
ProbeName

Please see example file in data/ directory

REQUIRED:
plate_bc
well_bc
OPTIONAL: additional columns with well metadata, column headers are user defined, e.g. drug_concentration
Please see example file in data/ directory

BLASTn

pandas

py-editdist

NumPy

1. H. Li, J. Qiu, X.-D. Fu, RASL-seq for massive parallel and quantitative analysis of gene expression, Curr. Protocol. Mol. Biol., 98 (2012), pp. 4.13.1–4.13.9

2. Larman HB, Scott ER, Wogan M, Oliveira G, Torkamani A, Schultz PG, Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay, Nucleic Acids Res. 2014;42(14):9146-57